Download 2010 Class Abstracts appear below with my editorial additions in red

2010 Class Abstracts appear below with my editorial additions in red. –ams
Clare Bruggeman-November 2, 2010
Luciferase Reporter Mycobacteriophages (LRP) Assays for Diagnosis of Antibiotic Resistant
Mycobacterium tuberculosis
The World Health Organization (WHO) reported an estimate of 9.9 million incident cases of
tuberculosis throughout the world in 2008, with as many as 2.3 million of those cases ending in death.
Tuberculosis is an infectious disease caused by the airborne pathogen, Mycobacterium tuberculosis, and
in spite of the dire statistics, the disease is curable. Early diagnosis is essential to saving the lives of
individual patients, preventing epidemic spread, and curtailing the evolution of multi-drug resistant
strains. The antibiotic regimen used to cure tuberculosis patients is long and requires simultaneous usage
of multiple antibiotics. Interruptions in the treatment (whether from patient noncompliance, or disruptions
in drug availability) lead to the development of multi-drug resistant strains of M. tuberculosis. Thirty
thousand cases of multi-drug resistant tuberculosis were reported in 2008. The traditional method of
detecting the tuberculosis bacilli, used for the last one hundred years, is called acid-fast bacilli smear
(AFB). More recently, other radiometric detection methods like MGIT BACTEC 960 were developed.
Although these techniques are faster, they are also more expensive, requiring lab facilities that are not
available in the areas of the world that are devastated by tuberculosis. In that light, scientists have been
working to develop luciferase reporter mycobacteriophage (LRP) assays to rapidly diagnose patients with
antibiotic resistant strains.
Although a luciferase assay for diagnosis of tuberculosis was already in existence, in 2001,
Banaiee et al. compared the MGIT BACTEC 960 methods with the luciferase assay for speed, sensitivity
and specificity. They concluded that with the proper precautions, the LRP method was comparable to
MGIT BACTEC 960 and actually provided the quickest turn-around on diagnosis of susceptibility to the
first-line antibiotics. The one major disadvantage of the LRP method is that depending on the species of
phage, some of these LFP assays are unable to discriminate between the nontuberculosis mycobacteria
(NTM) and the disease causing M. tuberculosis.
Kumar et al (2008) continued with the development of an LRP tuberculosis assay by screening
phage samples for an isolate with increased light production and the capability of infecting M.
tuberculosis. The Che12 phage was selected as a likely candidate and the firefly luciferase gene was
cloned into the Che12 phage. The transgenic Che12 phage was determined to be “ideally suited” to be
used as a diagnostic tool because of the increased sensitivity of this temperate phage as well as the
broader host range: Che 12 also infects the nonpathogenic M. smegmatis which is used in labs as the
mycobacterium model species.
References
WHO.int. 2010. The World Health Organization. 29 October 2010. <http://www.who.int/en>.
CDC.gov. 2010. The Center for Disease Control and Prevention. 29 October 2010.
<http://www.cdc.gov>.
“Hypersensitivity.” 2010. Practical Pathology Class Website. Department of Pathology, University of
Cambridge. 29 October 2010. <http://www.path.cam.ac.uk/partIB_pract/P09/>.
Kumar, V., P. Loganathan, G. Sivaramakrishnan, J. Kriakov, A. Dusthakeer, B. Subramanyam, J. Chan, W.
Jacobs Jr. and N. Paranji Rama. 2008. “Characterization of temperate phage Che 12 and construction of a
new tool for diagnosis of tuberculosis.” Tuberculosis 88, 616-623.
Banaiee, N., M. Bobadilla-del-Valle, S. Bardarov Jr., P. F. Riska, P. M. Small, A. Ponce-de-Leon, W.
Jacobs Jr., G. F Hatfull and J. Sifuentes-Osornio. 2001. “Luciferase Reporter Mycobacteriophages for
Detection, Identification, and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis in
Mexico.” J Clin Microbiol 39 (11), 3883-3888.
Use of Brilliant Blue G to Improve Recovery After a Spinal Cord Injury
Ethan Doerr November 2, 2010
Spinal cord injuries have been sustained throughout the existence of humans and are
becoming more of an issue due to our increased enjoyment of dangerous physical contact
activities such as sports. However, despite the increasing occurrence of these injuries, progress
on stopping and reversing damage has failed to occur. Medical miracles may not be far behind,
however, as researchers at the University of Rochester Medical Center have made significant
discoveries.
Research has shown that neuron death stems from an excess of extracellular ATP released by
damaged neurons in the spinal cord. This excess ATP reacts with the ATP-sensitive receptor
P2X7, which in turn changes the phenotypes of astrocytes within the neuron. This change in the
astrocytes causes the neuron to fire continuously, resulting in cell necrosis. Due to this,
undamaged neurons are also killed due to the original injury, resulting in the disruption of
nervous signals in the spinal cord. Without treatment, this may cause the subject to have limited
use of the body beyond the damage, or lose functionality all together.
In an attempt to combat the loss of functionality, antagonists to the P2X7 receptor was
administered after the spinal injury, in this experiment the antagonist being Brilliant Blue G
(BBG). This reacted with the P2X7 receptor, inhibiting the ability of the ATP to be taken up. In
turn, the astrocytes were not affected, which kept the cell from undergoing necrosis. Three
groups of lab rats were all subjected to the same spinal injury and then tested for hind leg
functionality, upon which paraplegia was observed in all subjects. BBG was then administered
in doses of 10 mg/kg and 50 mg/kg to groups one and two respectively, while the third group
was left as a control. Treatment commenced 15 minutes after injury and continued once a day
for three days.
Results of the test show the control subjects to have regained the ability of movement in the
hind legs, however not the ability to coordinate movement with the forelegs or the ability to
support walking. The subjects treated with the BBG showed marked improvement, ultimately
regaining the ability of coordinated movement and the ability to walk, however sometimes with a
noticeable limp.
References
Gandelman et al., Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron
death: implications for amyotrophic lateral sclerosis Journal of Neuroinflammation 2010, 7:33
Weiguo Peng, Maria L. Cotrina, Xiaoning Han, Hongmei Yu, Lane Bekar, Livnat Blum,
Takahiro Takano, Guo-Feng Tian, Steven A. Goldman, and Maiken Nedergaard. Systemic
administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after
spinal cord injury. PNAS (2009) 106(30): 12489–12493
Danielle Cervantes, November 4, 2010
The use of thymidine kinase serum in the prognosis of cancer and the effectiveness in
treatment of non-Hodgkin’s lymphoma
This presentation will discuss the use of thymidine kinase serum in the prognosis of
cancer and the effectiveness in treatment of non-Hodgkin’s lymphoma. Key definitions will be
addressed as well. A basic overview of the experiments involving thymidine kinase serum will
be given. Also, a brief explanation of new techniques will be given. Finally, the effectiveness of
thymidine kinase serum in the prognosis of cancer and the effectiveness of treatment will be
discussed. [more scientific detail would be welcome in this abstract]
References
Chen, Yan, Mingang Ying, YanSong Chen, Minhua Hu, Yingying Lin, Dedong Chen, Xiaoli Li,
Ming Zhang, Xia Yun, Ji Zhou, Ellen He, and Sven Skog. "Serum Thymidine Kinase 1
Correlates to Clinical Stages and Clinical Reactions and Monitors the Outcome of
Therapy of 1,247 Cancer Patients in Routine Clinical Settings." Int J Clin Oncol 15
(2010): 359-68. Print.
"NCI Dictionary of Cancer Terms." National Cancer Institute - Comprehensive Cancer
Information. Web. 02 Nov. 2010. <http://www.cancer.gov>.
“Dot Blot Assay Methods, Techniques, Protocols." Antibody Search Engine and Suppliers
Guide. 2007. Web. 01 Nov. 2010.
<http://www.antibodybeyond.com/applications/dotblot.htm>.
"Electrochemiluminescence (ECL) Assay." Critical Reagents Program. Web. 02 Nov. 2010.
<www.jpeocbd.osd.mil/packs/DocHandler.ashx?DocID=8597>.
"Non-Hodgkin Lymphoma Home Page - National Cancer Institute." National Cancer Institute Comprehensive Cancer Information. Web. 02 Nov. 2010.
<http://www.cancer.gov/cancertopics/types/non-hodgkin>.
Pan, Zhu-Lin, Xing-Ying Ji, Yan-Min Shi, Ji Zhou, Ellen He, and Sven Skog. "Serum
Thymidine Kinase 1 Concentration as a Prognostic Factor of Chemotherapy-treated NonHodgkin's Lymphoma Patients." J Cancer Res Clin Oncol 136 (2010): 1193-199. Print.
"ROC Curve Analysis: Introduction." MedCalc Statistical Software. 1993. Web. 02 Nov. 2010.
<http://www.medcalc.be/manual/roc.php>.
Engineered Tobacco As a Means for Biofuel
Lee Greenawald, November 4, 2010
About fifteen billion cigarettes are sold daily - or ten million every minute.
Researchers are now finding that tobacco plants, instead of being grown for toxic
applications can ironically be advantageous to the economy in a different way. Engineered
tobacco plants can generate a large amount of inexpensive biomass more efficiently than
almost any other agricultural crop grown. Tobacco is a very attractive biofuel because it is
ideal to choose plants that are not used in food production (like corn or soybeans). Tobacco
plants contain effective oil biosynthesis machinery and can contain up to 40% of seed
weight in oil per dry weight. Tobacco leaves contain 1.7%-4% oil per dry weight, which is
extractable as fatty acid esters, the major component of biofuel oil. Two techniques to over
express genes involved in oil production have been identified to increase the overall oil
content in tobacco leaves.
The first technique is based on the numerous studies of gene manipulation enabling
the elevation of oil storage in alternative plant organs (such as roots, stems or leaves)
making the green biomass an eligible system for manufacturing biodiesel. The Arabidopsis
thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in
triacylglycerol (TAG) biosynthesis in tobacco was overexpressed under the control of a
specific small subunit promoter. This modification led up to a 20-fold increase in TAG
accumulation in tobacco leave which translates into an overall twofold increase in extracted
fatty acids that is normally produced.
The second technique was expressing Arabidopsis gene LEAFY COTYLEDONE 2
(LEC2), which is a master regulator of seed maturation and seed oil storage under the
control of an inducible A/c promoter. This led to an accumulation of up to 6.8% dry weight
of total extracted fatty acids.
By overexpressing these specific genes in tobacco seeds, the overall quantity of oil
increases to hopefully create a mass production of tobacco oil as a platform for “energy
plants”. Manipulating metabolic pathways has proven to increase the oil accumulation in
tobacco by twofold. More research is being performed to increase oil content including
blockage of lipid breakdown or inhibition of the pathways that divert energy and metabolite
flow from oil biosynthesis.
References:
Andrianov, V.; Borisjuk, N.; Pogrebnyak, N.; Brinker, A.; Dixon, J.; Spitsin, S.; Flynn, J.;
Matyszczuk, P.; Andryszak, K.; Laurelli, M.; Golovkin, M.; Koprowski, H. Tobacco as
a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2
genes increases accumulation and shifts the composition of lipids in green biomass.
Plant Biotechnology Journal. 2010, 8,277-287.
Engineered tobacco plants have potential as biofuel feedstock; expressing oil in the leaves.
http://www.greencarcongress.com/2009/12/tobacco-20091231/html. (accessed
November 1, 2010).
Luo, K.; Duan, H.; Zhao, D.; Zheng, X.; Deng, W.; Chen, Y.; Stewart, N.; McAvoy, R.;
Jiang, X.; Wu, Y.; He, A.; Pei, Y.; Li, Y. Plant Biotechnology Journal. 2007, 5, 263274.
Stricklen, M. Plant genetic engineering to improve biomass characteristics for biofuels.
Current Opinion in Biotechnology. 2006, 17, 315-319.
Usta, N. Use of tobacco seed oil methyl ester in a turbocharged indirect injection diesel
engine. Biomass and Bioenergy. 2005, 28, 77-86.
Wu, S.; Chappel, J. Metabolic engineering of natural products in plants; tools of the trade
and challenges for the future. Current Opinion in Biotechnology. 2008, 19, 145-152.
Production of biologically active human GM-CSF in the seeds of transgenic
rice plants
Katie Surckla, November 4, 2010
Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a
cytokine which regulates production and function of white blood cells which are
essential for fighting infections. A mature hGM-CSF is a polypeptide and is a part of
clinical management for life-threatening neutropenia. Neutropenia is the most
common toxicity of cancer chemotherapy. Other possible pharmaceutical
applications could be for patients with pneumonia, Crohn’s fistulas, diabetic foot
infections and a variety of other infectious conditions including HIV-related
opportunistic infections.
Therapeutic recombinant proteins are produced in mammalian cells or single
celled organisms like yeast and bacteria. These methods are very costly, so
producing recombinant protein-based pharmaceuticals in green plants is being
explored as a cheaper method. The production of GM-CSF has also been expressed
in tobacco and sugarcane, but in really low levels. Rice seeds have proven to be the
more effective plant for higher levels of expression of hGM-CSF. Standard DNA
cloning and DNA amplification techniques were followed in producing these
transgenic rice plants. Agrobacterium was transformed with the binary vector
containing the GM-CSF construct. The plants were then cultured and grown. To
verify the integration of GM-CSF protein, the rice genomic DNA was isolated and
purified. PCR and Southern blotting were used along with a 32P-probe containing the
hGM-CSF sequence. Then, to test specifically for GM-CSF, ELISA and Western
blotting techniques were used. Western blot showed a positive detection at 18 kDa
(the molecular weight of hGM-CSF, non-glycosylated).
After analysis and verification of hGM-CSF in the transgene rice plants, the
rice seeds were found to contain 1.3% of the total soluble protein which is 4-fold
higher than the reported expression level in the seeds of tobacco. The use of rice as
a host further proves to be beneficial because it is a safe production in seeds, but it
is also self-containment of foreign genes, meaning that rice is a self-pollinated crop
plant so there won’t be any cross-contamination. Production of these transgenic
rice crops can help in the pharmaceutical industry and help bring down the cost of
this helpful protein.
References:
1) Sardana, Ravinder. et. al. 2007. Biologically Active Human GM-CSF Produced in
the Seeds of Transgenic Rice Plants. Transgenic Research. 16: 713-721.
2) Wang, Ming-Li. et. al. 2004. Production of Biologically Active GM-CSF in
sugarcane: a secure biofactory. Transgenic Research. 14: 167-178.
3) Blais, David R. et. al. 2007.Humanizing infant milk formula to decrease postnatal
HIV transmission. TRENDS in Biotechnology. 25(9): 376-384.
Genetic engineering of “golden rice”, Production of beta carotene in rice endosperm
Philip Nam-Nov 4th, 2010
Golden rice was developed to provide a low cost vitamin supplement in countries with
individuals suffering from vitamin A deficiency. Vitamin A deficiency or VAD is responsible for
around one to two million deaths worldwide with an additional five hundred thousand cases of
blindness caused by VAD, fifty percent of individuals who became blind died within a year. VAD
also impacts the immune system leaving effected people susceptible to a variety of diseases.
Children and pregnant woman have the highest risk of developing VAD. Vitamin A
supplementation programs take place internationally through pills and injections however by
producing rice, a primary food source in many of the countries affected, that expresses beta
carotene. A more readily available and steady low dose regimen of Vitamin A could be created
that could be afforded by even the poorest sectors of developing countries.
Golden rice was designed to produce beta carotene, a precursor to vitamin A. Psy
(phytoene synthase) from daffodil (Narcissus pseudo narcissus) and Crt1 from the soil
bacterium Erwinia uredovora catalyze the biosynthesis of beta-carotene from geranyl
diphosphate. Beta-carotene is assumed to be converted to retinal and subsequently vitamin A
in the animal gut. The insertion of a lyc (lycopene cyclase) gene was thought to be needed, but
research showed it was already being produced in wild-type rice endosperm. The gene
construct used to generate golden rice included Glu, rice endosperm-specific glutelin promoter
to ensure that the gene was only expressed in the endosperm of the rice. tpSSU, a small subunit
transit peptide for chloroplast localization. Nos, as the terminator. The Psy, gene from Narcissus
pseudo narcissus, (GR1) or Zea maize (GR2), Ubi1, maize polyubiquitin promoter. Pmi,
phosphomannose isomerase gene from E. coli for positive selection. The original golden rice
was called SGR1, and under greenhouse conditions it produced 1.6 µg/g of carotenoids.
In 2005 researchers at the biotechnology company Syngenta. Created a variation on golden rice
they named "golden rice 2". They combined the phytoene synthase gene from maize with crt1
from the original golden rice. Golden rice 2 produces 23 times more carotenoids than golden
rice.
References
Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin
A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:
303-305.
the science behind golden rice <http://www.goldenrice.org/Content2-How/how1_sci.html>
the golden rice scandal unfolds <http://www.i-sis.org.uk/goldenRiceScandal.php>
Plantibodies: Producing Human Immunoglobulin G in Plants
Amanda Forni, November 4, 2010
The use of plants as hosts to produce antibodies for medical use has become very
advantageous in recent years. The ability to use both Agrobacterium-mediated transformation
and particle bombardment allows for diversity and selectivity of the antibodies. Through the
production of antibodies in plants, the transgene expression is able to be stabilized throughout
generations of plants and through the use of viral vectors or agroinfiltration, a large amount of
product can be produced within weeks. While the glycosylation of the antibodies is different
between humans and plants, researchers have humanized a plant glycan in tobacco by
introducing human β-1,4-galactosyltransferase. Consequently, the tobacco plant is a common
bioreactor that is used.
A study done by a group of researchers in Italy compared the production of human
immunoglobulin G in plants using stable transgenic lines and a transient expression system. In
the first trial, the IgG genes were cloned into a plant binary vector with a 35S CaMV promoter
and an omega translation enhancer sequence. Multiple transgenic plants were screened and the
best selected for cross pollination. The offspring were grown, tested for the antibody, and then
purified. In the second trial, Agrobacterium tumefaciens contained the cloned IgG and infected
the N. bethamiana plant. Leaves were collected and tested for the antibody each day, with 6
days after infiltration containing the highest expression levels.
The results showed that stable transgenics require a long time to produce low quantities
of product, while transient expression occurs quickly with a much larger quantity of the protein.
References:
Sharma, A. K., Sharma, M. K. 2009. Plants as bioreactors: Recent developments and emerging
opportunities. Biotechnology Advances. 27: 811-832.
Stoger, E., Sack, M., Fischer, R., Christou, P. 2002. Plantibodies: application, advantages and
bottlenecks. Current Opinion in Biotechnology. 13: 161-166.
Villani, M. E., Morgun, B., Brunetti, P., Marusic, C., Lombardi, R., Pisoni, I., Bacci, C.,
Desiderio, A., Benvenuto, E., Donini, M. 2009. Plant pharming of a full-sized, tumour-targeting
antibody using different expression strategies. Plant Biotechnology Journal. 7: 59-71.
Dawn
Recombinant Baculovirus: A Potential Vector for Gene Therapy of Cancer
Jenna Kausner, November 4, 2010
Modern cancer therapeutics, such as surgical resection of the primary tumor or chemotherapy,
are often inefficient due to their inability to control metastasized tumors and promote long-term survival.
Essentially, most cancer is incurable with these conventional methods. In these cases, gene therapy has
been proposed as a possible solution to conventional cancer therapeutics by using it either in
combination with conventional cancer therapeutics or as a replacement to these old methods. The
general goal of cancer gene therapy is to introduce foreign therapeutic genes into cancer cells that result
in temporary suppression or complete eradication of the tumor. There have been several different
approaches to cancer gene therapy, including immunogene therapy such as cytokine gene transfer,
selective prodrug activation (“suicide genes”), tumor suppressor gene transfer, and antisense techniques
to inhibit activated oncogenes. There are certain basic requirements to consider in order for gene therapy
to be successful: safety, efficiency, specificity, expression level of transduced gene, and simplicity of
manufacture. Depending on the therapeutic strategy and type of cancer, these requirements can be
difficult to satisfy. The two main methods of delivery for cancer gene therapy are DNA/RNA viral vectors
and non-viral vectors. One of the increasingly popular methods is using mammalian viral-based cancer
gene therapy; however, the efficiency has been fairly low in clinical trials and the safety is questionable.
Other research groups have focused on using replication-competent oncolytic virus, but they tend to
perpetually exist in mammalian cells, which is a safety concern. In Paul et al. (2010), they evaluated the
insect-cell specific baculovirus as a vector for gene delivery to colorectal cancer cells in addition to other
cancer types such as breast, pancreas, and brain.
The use of baculovirus as a vector to target tumor cells can be a powerful took tool due to the fact
that it will enter mammalian cells but cannot replicate, increasing efficiency and eliminating safety
concerns with previous vectors (the natural virus promoters are not active in mammalian cells). A
recombinant baculovirus was constructed with the Monster Green Fluorescent Protein (MGFP) reporter
gene and the human cytomegalovirus (CMV) immediate-early promoter. This construct is known as the
BacMam virus because now the insect baculovirus can express its transgene in mammalian cells. In their
experiment, Paul et al. took human colon cancer cells (SW480), breast cancer cells (SkBr3),
neuroblastoma cancer cells (Neuro2A), hepatocellular carcinoma cells (HepG2), and pancreatic
carcinoma cells (PANC-1) and maintained them on appropriate mediums with supplemented with 10%
Fetal Bovine Serum (FBS) as stationary cultures. Spodoptera frugiperda (Sf9) insect cells were
maintained in a medium without FBS. These Sf9 cells were maintained in exponential growth phase and
subcultured twice per week. The MGFP gene driven by the CMV promoter was cloned into the
baculovirus transfer vector and this recombinant transfer vector was amplified in E. coli. Recombinant
baculoviruses were generated using the Sf9 insect cells. Then, SW480 cells were transduced with
baculovirus in 96-well plates. MGFP protein expression was detected by extracting total RNA and
performing RT-PCR using specific primers for the MGFP gene. The expression of MGFP was
successfully detected in transduced SW480 cells. Transduction efficiency was much higher in SW480
cells than any of the other cancer cells, but similar in HepG2 cells. Leukemic cells showed poor
transduction efficiency. Although high transduction efficiency was observed with SW480 cells, this
gradually diminished due to the fact that baculovirus does not naturally integrate with the host organism,
so long-term expression is not observed. This is important in terms of safety because the vector won’t
randomly integrate into crucial gene regions and cause unexpected tumerogenesis, whereas mammalian
viral vectors would. However, transient expression can be problematic and can possibly be solved by
using a hybrid baculovirus-adeno associated viral vector for more prolonged expression. In addition,
multiple administrations of the viral dose may also overcome transient expression.
References:
Paul, A.; Jardin, B. A.; Kulamarva, A.; Malhotra, M.; Elias, C, B.; Prakash, S. Recombinant Baculovirus as a
Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning,
Expression, and In Vitro Characterization. Mol Biotechnol [Online] 2010, 45, 129 ff.
Zhang, J.; Russell, F. J. Vectors for cancer gene therapy. Cancer and Metastasis Reviews [Online] 1996,
15, 385 ff.
Gunji, Y.; Ochiai, T.; Shimada, H., Matsubara, H. Gene Therapy for Cancer. Surg Today [Online] 2000, 30, 967 ff.
Marathon Mice (a more informative title would be nice)
Joellen Little-Kemper, November 9, 2010
What if the enhancement of a single gene could give a person the benefits of exercise without
requiring them to lift a finger? Researchers at the Howard Hughes Medical Institute and the Salk
Institute have found just that. They were able to pinpoint a transcription factor that, once enhanced,
could control obesity resistance, insulin sensitivity, muscle fiber specification, and physical endurance.
The gene of significance here is the PPAR-delta (PPAR standing for peroxisome proliferator-activated
receptor), which was fused to the 78-amino acid transactivation domain of VP16, and under the control
of a human a-skeletal actin promoter. This transgene was injected into zygotes. The transgenic mice
were compared against their wild type littermate controls in a series of experiments. One of these
experiments included the transgenic and wild type, of approximately the same body weight, being fed a
high fat diet and the percentage of weight gain for the two types of mice were compared. This high-fatinduced-obesity experiment was also used to monitor the histology of inguinal fat, the intramuscular
glycogen and triglyceride content, and glucose tolerance. In a separate test, the two groups were also
compared for exercise performance on a special treadmill. Muscle fiber typing of the two groups also
took place, which involved metachromatic dye-ATPase methods and analyzation of the mitochrondial
composition.
The PPAR-delta was found to increase the myoglobin and mitochondrial content, which then
contributed to the muscle fiber switching to type I muscle fibers. After experimentation with the high fat
diet, the transgenic mice displayed significantly higher oxygen consumption; the wild type controls
became obese, whereas the transgenic stayed at a normal body weight/fat composition; the transgenic
showed smaller adipocyte cell size and decreased triglyceride content. While experimenting for physical
performance, the transgenic mice were able to run on an oxygen-infused, enclosed treadmill almost 1
hour longer – equating to almost a kilometer further than their wild type counterpart.
From these results, the researchers have concluded that it could be possible to develop a drug
which produces all of the same benefits. Four years after the initial findings were published, the same
team of scientists were focused in on finding a drug that was specific for the PPAR delta. They found this
in an investigational drug identified as GW1516. Results from experimentation with this drug concluded
that it only enhances performance when combined with a regular exercise regimen – which is great for
athletic competitors, but not so much for those who want the benefits of exercise without all of the
work. We should not lose hope though, with the ever increasing medical advances it would not be
surprising to one day get our daily dose of exercise in the form of a little pill.
Sources used:
Wang Y-X, Zhang C-L, Yu RT, Cho HK, Nelson MC, et al. (2004) Regulation of Muscle Fiber Type and
Running Endurance by PPARδ. PLoS Biol 2(10): e294. doi:10.1371/journal.pbio.0020294
"Salk Institute - Press Releases - Exercise in a Pill." Salk Institute | Where Cures Begin. Web. 02 Nov.
2010. <http://www.salk.edu/news/pressrelease_details.php?press_id=306>.
"Marathon Mouse." The Molecular Biology of Paradise. Web. 02 Nov. 2010. <http://www.paradiseengineering.com/somatic/index.html>.
Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA + Multiplex
amplification of the mammoth mitochondrial genome and the evolution of Elephantidae
Peter Malinski, November 9, 2010
Mammoths are an extinct species that lived during the Pleistocene as recently as 10,000 years
ago. Some mammoths have been recovered in remarkable condition in recent times which has led to
scientific inquiry about the genetics and phylogeny of this creature. Even though ancient DNA has a
plethora of inherent problems in replication, this scientific inquiry has advanced to the point of
reproducing this creature’s entire mitochondrial genome.
The first paper by Briggs et al. deals with one of the many common problems associated with
ancient DNA (ancient DNA being defined as DNA samples from ancient organisms), which is higher
error rates primarily due to uracil bases created by cytosine deamination. The authors attempted to
remove the uracil residues and also repair most of the resulting abasic sites by utilizing a regime of
Uracil-DNA-Glycosylase and endonuclease VIII. The technique used by the authors is called Highthroughput direct sequencing (which sequences DNA molecules of all lengths) where the ends of ancient
DNA fragments are made amenable to ligation by treatment with T4 DNA polymerase and T4
polynucleotide kinase. In theory this technique would allow better results than a direct PCR since
deamination of cytosines could occur in conjunction, and as a result they were successful in the
deamination process.
The second paper by Krause et. al. deals with multiplex PCR which allowed the entire
mitochondrial genome to be amplified using just two initial amplifications accompanied by primer pairs
that defined overlapping DNA sequence fragments. With multiple PCR amplification and 200mg of
mammoth bones the entire mitochondrial genome was sequenced which allowed for determination of the
phylogenic relationship of the wooly mammoth to the Asian elephant as well as to the African elephant.
Sources:
1. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae.
Krause J, Dear P, Pollack J, Slatkin M, Spriggs H, Barnes I, Lister A, Ebersberger I, Paabo S, Hofreiter
M. Nautre Vol 439 9 Feb. 2006 doi: 10.1038/nature04432
2. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Briggs A,
Stenzel U, Meyer M, Drause J, Kircher M, and Paabo S. Nucleic Acids Research, 2010, Vol. 38, No 6. 22
December 2009.
3. Orlando L, Darlu P, Toussaint M, Bonjean D, Otte M, and Hanni C. (2006) Revisiting Neandertal
diversity with a 100,000 year old mtDNA sequence. Curr Iol., 16,
4. Paabo S, et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645-679 (2004).
5. Tajima F. Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135, 599607 (1993)
6. Felsenstein, J. Confidence-limits on phylogenies with a molecular clock. Syst. Zool. 34, 152-161
(1985)
Need to include page numbers in the first three sources/references that you list. All sources
should be cited using a single citation style.
Ryan Gertz
PBIOS abstract
Disease Phenotype of a Ferret CFTR-Knockout Model of Cystic Fibrosis
This article was on using ferrets as a model for the genetic disease cystic fibrosis. Models
for this disease already exist as well as for many other genetic diseases. Mice and pigs have been
used to model cystic fibrosis, but ferrets were carefully considered due to the similarity of their
lung anatomy to that of humans. Since lung complications are the leading causes of fatality in
cystic fibrosis patients, it stands to reason that a model similar to humans would be chosen.
Models for genetic diseases are a nice commodity to engage with because they can show the
likely progression of a disease. In other words, it can be used to study a disease without the
ethical complications of using human patients.
Since ferrets have not been used to model cystic fibrosis before, a baseline of data for the
disease is needed, and this study looks at that. Cystic fibrosis leads to complications in the
intestines, the pancreas, the liver, sweat glands, gall bladder, and even leads to infertility in
males. These different organs and tissues were looked at and compared with other models of the
disease leading to interesting conclusions and specific reasons for using one model over another
in certain tissues.
The ferrets used were knockout gene models, meaning they were engineered using
Mundelein inheritance and somatic cell nuclear transfer to transform the parent ferrets. Since
Cystic Fibrosis is caused by a mutation in the CFTR gene, this was the gene that was knocked
out. When these models were born, they immediately had complications to overcome, such as
intestine blockage, or acute lung infection. The other affected tissues did not show complications
that needed as immediate attention. Overall, this was a great study to get a baseline for using
ferrets as a CF model, and further studies are needed to see if they were a good choice for
modeling the disease progression in human lungs.
References:
1.) Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis Published in
Volume 120, Issue 9 (September 1, 2010)
J Clin Invest. 2010;120(9):3149–3160. doi:10.1172/JCI43052.
Copyright © 2010, American Society for Clinical Investigation
2.) Riordan JR, et al. Identification of the cystic fibrosis gene: cloning and characterization of
complementary DNA. Science. 1989;245(4922):1066–1073.
3.) Egan ME. How useful are cystic fibrosis mouse models? Drug Discov Today Dis Models.
2009;6(2):35–41.
4.) Rogers CS, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in
newborn pigs. Science. 2008;321(5897):1837–1841.
Enviropigs (a more informative title would be nice, e.g. Genetic engineering of the Enviropig to
reduce phosphorous content in swine manure)
Kristen Greer, November 9, 2010
Swine manure can be an excellent phosphorous fertilizer, but can cause a build up of
phosphorous in the soil. Pigs are fed corn, soybeans, barley, and other cereal grains in which
contain an indigestible compound called phytate. Approximately 50 to 75% of the phosphorous
present in these cereal grains is phytate. The phytate present in the manure is about four times
greater due to the proteins and carbohydrates in the cereal grains being digested and absorbed.
Runoff of the high phosphate content soil in areas where swine production is high into ponds,
streams, rivers and lakes can allow extensive algal growth which can lead to anoxia in fresh
water systems and can even increase the growth of blue green algae which often leads to the
production of toxins
Scientists have genetically modified a line of Yorkshire pigs to produce phytase in the
salivary glands to secrete the enzyme in the pig’s saliva which breaks down the phytate present
in the pig’s diet. This allows us to increase food production without degrading the environment.
To get these pigs to secrete the phytase in their saliva, scientists have constructed a
transgene consisting of a murine (mouse) parotid secretory promoter gene sequence and the E.
coli phytase gene and was introduced into the pig’s chromosomes inside of a fertilized pig
embryo by microinjection. The fertilized egg was then introduced into a sow’s reproductive
tract. The results were that 33 transgenic founder piglets were produced and 14 of them produced
phytase in their saliva after about 7-11 days of age. The phytate phosphorous content in the
manure of G1 piglets was reduced by up to 75%. This line of pigs is now in its 8th generation and
enviropigs are waiting to be approved by the FDA for consumption.
References:
Golovon, S. et al. (2001). Pigs expressing salivary phytase produce low-phosphorous manure.
Nature Biotechnology, 19. (need to provide page numbers) Retrieved from
http://biotech.nature.com
Enviropig. (2010). Informally published manuscript, Technology, University of Guelph, Ontario,
Canada. Retrieved from http://www.uoguelph.ca/enviropig/technology.shtml
Jason Gregorin
PBIO 450
GFP Abstract
Green fluorescent protein is a common protein used for various methods of cellular study. It is a
fairly short peptide sequence, consisting of 238 amino acids. Its usefulness comes from its size, its
natural fluorescence without outside chemicals, and its ability to be attached to proteins for qualitative
and quantitative measurements. There also exists various other fluorescent proteins such as red
fluorescent protein.
In nuclear-cytoplasmic dynamics, GFP and RFP are utilized in order to view cellular changes on a
real-time basis. GFP is linked to a histone protein, causing GFP to only be produced in the nucleus of
cells. RFP is encoded to be produced in the cytoplasm of cells. This allows for imaging of changes in the
size and shape of the nucleus and cytoplasm of cells as they progress through the cell cycle. This
technique can also be utilized to further understand the changes that occur in cancer and apoptosis.
Further progression with this technique can lead to a greater understanding of cell functions as well as
give a tool that allows for easier testing of cancer treatments.
References
HOFFMAN, R. (2008). In vivo real-time imaging of nuclear-cytoplasmic dynamics of dormancy,
proliferation and death of cancer cells. APMIS, 116(7/8), 716-729. doi:10.1111/j.16000463.2008.01036.x.
Haldar, S., & Chattopadhyay, A. (2009). Green fluorescent protein: a molecular lantern that
illuminates the cellular interior. Journal of Biosciences, 34(2), 169-172. Retrieved from Academic
Search Complete database.
The time of appearance of Lewy Body Inclusions and the effect of coffee and decaffeinated
extracts on Drosophila neuronal cultures.
Teng Zhang. Nov/07/2010
Lewy Bodies are amyloid, proteinacous aggregations mainly composed of α-synuclein.
They are one of the hallmarks of Parkinson’s disease which causes the death of dopaminergic
neurons in the Substantia Nigra Pars Compacta region of the brain. Despite Although Lewy
Bodies are well defined, information on their sizes and time of appearances are absent in the
scientific literatures. In my presentation, I present my recent results to clearly define the time of
appearance of Lewy Bodies in a Drosophila melanogaster model (of what?) and investigate the
effects of known neuro-protective drugs such as coffee and tobacco extracts. The Drosophila
model I used expresses the mutant form of α-synuclein, A53T, which increases the propensity of
Lewy body aggregation. Cells were obtained from Drosophila eggs and differentiated in to
neuron cells and drugs are added at three days in vitro and left to grow in media for six and nine
days in vitro before staining with α-synuclein monoclonal antibodies and corresponding
secondary antibodies. From my data, I found out that α-synuclein is unregulated early in
neuronal cultures and that the ratio of dopaminergic neurons to Lewy bodies stays constant
inferring that Lewy body is a rescue (result of?) rather than the cause. Finally, my results show
that there is a possibility that caffeine alone rescues dopaminergic neurons from Lewy Bodies.
[Note this presentation is supposed to be about a primary research paper, not your own work.]
References:
O’Reilly EJ, McCullough ML, Chao A, Henley SJ, Calle EE, Thun MJ, Ascherio A (2005)
Smokeless tobacco use and the risk of Parkinson’s disease mortality. Mov Disord 20:1383–
1384.
Kien Trinh. Laurie Andrews, James Krause, Tyler Hanak, Daewoo Lee, Michael Gelb, and Leo
Pallanck. Decaffeinated Coffee and Nicotine-Free Tobacco Provide Neuroprotection in
Drosophila Models of Parkinson’sDisease through an NRF2-Dependent Mechanism. The
Journal of Neuroscience. You need a year, volume number and page numbers.
Soon S. Park and Daewoo Lee. Selective loss of dopaminergic neurons and formation of Lewy
body-like aggregations in a-synuclein transgenic fly neuronal cultures. European Journal of
Neuroscience, Vol. 23, pp. 2908–2914, 2006.
Li Rebekah Feng, Howard J. Federoff, Stefano Vicini and Kathleen A. Maguire-Zeiss. (2010) Synuclein mediates alterations in membrane conductance: a potential role for synuclein
oligomers in cell vulnerability. European Journal of Neuroscience, Vol. 32, pp. 10–17.
Jennifer